The active site of [Fe Fe] hydrogenase contains a catalytic binuclear iron subsite coordinated by CN- and CO ligands as well as a unique azadithiolate (adt(2-)) bridging ligand. It has been established that this binuclear cofactor is synthesized and assembled by three maturation proteins HydE, -F, and -G. By means of in vitro maturation in the presence of N-15- and C-13-labeled tyrosine it has been shown that the CN- and CO ligands originate from tyrosine. The source of the bridging adt(2-)ligand, however, remains unknown. In order to identify the nitrogen of the bridging amine using HYSCORE spectroscopy and distinguish its spectroscopic signature from that of the CN- nitrogens, we studied three isotope-labeled variants of the H-cluster (N-15-adt(2-)/c(14)N(-), N-15-adt(2-)/(CN-)-N-15, and N-14-adt(2-)/(CN-)-N-15) and extracted accurate values of the hyperfine and quadrupole couplings of both CN- and adt(2-) nitrogens. This will allow an evaluation of isotopologues of the H-cluster generated by in vitro bioassembly in the presence of various N-15-labeled potential precursors as possible sources of the bridging ligand.